Bisulfite conversion of dna

ABSTRACT

The present invention provides an improved method for the bisulfite conversion of DNA, and facilitates the analysis of cytosine methylation of genomic DNA. Novel combinations of denaturing solvents, new reaction conditions and new purification methods provide surprisingly efficacious methods for bisulfite conversion of DNA relative to prior art methods. The converted DNA may subsequently be analyzed by many different methods.

CROSS REFERENCE TO RELATED APPLICATION(S)

This application is a divisional of U.S. patent application Ser. No.10/964,167 filed Oct. 12, 2004, currently pending, which claims thebenefit of priority to German documents DE 103 47 396.3, DE 103 47397.1, DE 103 47 400.5 and DE 103 47 399.8 (all filed on 9 Oct. 2003),and also to U.S. patent application Ser. No. 10/311,661 entitled METHODSFOR DETECTING CYTOSINE METHYLATIONS, filed 18 Dec. 2002, based onPCT/DE01/02274, filed 19 Jun. 2001 and claiming priority to Germandocument DE 100 29 915.6, filed 19 Jun. 2000, all 10 of the forgoingpriority documents being incorporated by reference herein in theirentirety.

FIELD OF THE INVENTION

The present invention relates to methods for the detection of cytosinemethylation in DNA.

BACKGROUND OF THE INVENTION

5-Methylcytosine is the most frequent covalently modified base in theDNA of eukaryotic cells. For example, it plays a role in the regulationof transcription, in genetic imprinting and in tumorigenesis (or reviewsee Millar et al.: Five not four: History and significance of the fifthbase: in S. Beck and A. Olek, eds.: The Epigenome; Wiley-VCH VerlagWeinheim 2003, S. 3-20). The identification of 5-methylcytosine as acomponent of genetic information is thus of considerable interest.5-Methylcytosine positions, however, cannot be identified by sequencing,because 5-methylcytosine has the same base-pairing behavior as cytosine.Additionally, in the case of a PCR amplification, the epigeneticinformation borne by 5-methylcytosines is completely lost.

The usual methods for methylation analysis operate essentially accordingto two different principles. Either methylation-specific restrictionenzymes are utilized, or a selective chemical conversion of unmethylatedcytosines to uracil is conducted (bisulfite treatment). Theenzymatically or chemically pretreated DNA is then amplified and can beanalyzed in different ways (or review see Fraga and Esteller: DNAMethylation: A Profile of Methods and Applications:Biotechniques33:632-649, September 2002; and see also WO 02/072880 pp. 1 ff).

As the use of methylation-specific enzymes is restricted to certainsequences containing restriction sites recognized by said enzymes, formost applications a bisulfite treatment is conducted for review see U.S.patent application Ser. No. 10/311,661).

According to the invention described herein below, a “bisulfitereaction”, “bisulfite treatment” or “bisulfite method” refers to areaction for the conversion of cytosine bases in a nucleic acid touracil bases in the presence of bisulfite ions, whereby5-methyl-cytosine bases are not significantly converted. The bisulfitereaction contains a deamination step and a desulfonation step which canbe conducted separately or simultaneously (further details aredescribed, and a reaction scheme is shown in EP 1394172A1, incorporatedby reference herein in its entirety). There are various documentsaddressing specific aspects of the bisulfite reaction, including Hayatsuet al., Biochemistry 9:2858-28659, 1970; Slae and Shapiro, J. Org. Chem.43:4197-4200, 1978; Paulin et al., Nucl. Acids Res. 26:5009-5010, 1998;Raizis et al., Anal Biochem. 226:161-1666, 1995; and Wang et al. NucleicAcids Res. 8:4777-4790, 1980, and these documents, summarized in EP1394172A1 are also incorporated by reference herein in their entirety.

The bisulfite treatment is usually conducted in the following way: Thegenomic DNA is isolated, mechanically or enzymatically fragmented,denaturated by NaOH, converted several hours by a concentrated bisulfitesolution and finally desulfonated and desalted (e.g.: Frommer et al.: Agenomic sequencing protocol that yields a positive display of5-methylcytosine residues in individual DNA strands. Proc Natl Acad SciUSA. 89:1827-31, 1992, incorporated by reference herein in itsentirety).

In recent times several technical improvements of the bisulfite methodswere developed. The agarose bead method incorporates the DNA to beinvestigated in an agarose matrix, through which diffusion andrenaturation of the DNA is prevented (bisulfite reacts only onsingle-stranded DNA) and all precipitation and purification steps arereplaced by rapid dialysis (Olek A. et al. A modified and improvedmethod for bisulphite based cytosine methylation analysis, Nucl. AcidsRes. 24:5064-5066, 1996). In the patent application WO 01/98528 (=DE 10029 915; =U.S. application Ser. No. 10/311,661), a bisulfite conversionis described in which the DNA sample is incubated with a bisulfitesolution of a concentration range between 0.1 mol/l to 6 mol/l inpresence of a denaturing reagent and/or solvent and at least onescavenger. In said patent application several suitable denaturingreagents and scavengers are described (document incorporated byreference herein in its entirety). In the patent application WO03/038121 (=DE 101 54 317; =Ser. No. 10/416,624) a method is disclosedin which the DNA to be analysed is bound to a solid surface during thebisulfite treatment. Consequently, purification and washing steps arefacilitated. Further improvement are described in the patentapplications EP1394173A1 and EP1394172A1 (incorporated by referenceherein in its entirety).

However, a basic problem of the bisulfite treatment consists of the factthat long reaction times are necessary in order to assure a completeconversion and to exclude false-positive results. Simultaneously,however, this leads to a degradation of the DNA due to the long reactiontimes. Higher reaction temperatures in fact lead to a higher conversionrate, but also to a more intense degradation of the DNA. Theinteractions between temperature, reaction time, rates of conversion anddegradation were recently investigated systematically. In this way, itcould be shown that the highest conversion rates were attained attemperatures of 55° C. (with reaction times between 4 and 18 hours) andat 95° C. (with a reaction time of one hour). A serious problem,however, is the degradation of the DNA during this procedure. At areaction temperature of 55° C., 84-96% of DNA is decomposed. At 95° C.the degradation is in fact even higher (Grunau et al.: Bisulfite genomicsequencing: systematic investigation of critical experimentalparameters. Nucleic Acids Res. 29:E65-5, 2001; incorporated by referenceherein in its entirety). Thus, most authors use reaction temperatures ofapproximately 50° C. (see Frommer et al., loc. cit. 1992, p. 1827; Oleket al., loc. cit. 1996, p. 5065; Raizis et al: A bisulfite method of5-methylcytosine mapping that minimizes template degradation, AnalBiochem 226:161-6, 162, 1995).

In addition to the high degradation rate of DNA, there is anotherproblem in conventional bisulfite methods, which consists of the factthat a powerful purification method for converted DNA has not yet beendescribed. Many authors use precipitations (see Grunau et al., oc. cit).A purification via DNA-binding surfaces has also been described (seeKawakami et al.: Hypermethylated APC DNA in plasma and prognosis ofpatients with esophageal adenocarcinoma, Journal of the National CancerInstitute, 92:1805-11, 2000). The yield of these purifications, however,is limited.

Due to the high losses of the conventional bisulfite treatment, it isproblematic to use these methods for investigations in which thequantity of DNA to be analyzed is limited. A particularly interestingfield of methylation analysis, however, lies in diagnosing cancerdiseases or other disorders associated with a change in methylationstatus by means of analysis of DNA from bodily fluids, e.g. from bloodor urine. However, DNA is present only in small concentrations in bodyfluids, so that the applicability of methylation analysis is limited bythe low yield of conventional bisulfite treatment.

Accordingly, based on the particular importance of cytosine methylationanalysis and based on the described disadvantages of conventionalmethodology, there is a great technical need for improved methods ofbisulfite conversion.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the result of Example 1. The gel pattern of a PCR-amplifiedbisulfite-treated DNA strand is shown (left: molecular weight marker,right: PCR product).

FIG. 2 shows the results of Example 3 (use of DME). DNA was isolatedfrom plasma samples, treated with bisulfite and amplified by means of aLightcycler PCR. The Y-axis shows the fluorescent signal measured ineach cycle. The X-axis indicates the number of cycles. The curves of themethod according to the invention are shown on the left and those of theconventional method are shown on the right. It is shown that theoptimized method produces a significant fluorescent signal, even with asmall number of cycles. The DNA yield is higher than with theconventional method.

FIG. 3 shows the results of Example 4. The resulting gel from aelectrophoresis after a PCR amplification is shown. Two differentbisulfite-specific fragments, two nonspecific fragments and one genomicfragment were amplified each time. The uppermost panel shows the zerovalue (reaction time=0 hours). The second panel from the top shows thereaction with one thermospike and for one hour of total reaction time.The third panel from the top corresponds to the reaction with twothermospikes and two hours of total reaction time. The lowermost panelshows a reaction with 5 three thermospikes and three hours of reactiontime. A large part of the DNA is converted even after one hour with themethod according to the invention (second panel from the top). At thelatest after three hours, genomic DNA can no longer be detected(lowermost panel).

FIG. 4 shows the results of Example 5. The gel electrophoresis is shownafter a PCR amplification. Two different bisulfite-specific amplificateswere amplified. The panels on the left show conventional bisulfitetreatment, while those on the right show the method according to theinvention. A reaction time of 3 h is shown on the top and a reactiontime of 5 h is shown on the bottom. A clearly more sensitive detectionis possible with the method according to the invention (thermospikes).For example therefore, the fragment shown in the left lane can bedetected after 3 hours of reaction time, whereas the fragment from theconventional method still cannot be detected even after 5 hours ofincubation.

SUMMARY OF THE INVENTION

According to the present invention, addition of certain denaturingsolvents increases the conversion rate of the bisulfite reaction in anunexpected, surprising way. Simultaneously, the necessary reaction timeand consequently the degradation rate are reduced. According to thepresent invention, the denaturing solvents disclosed herein provideunexpectedly strong and advantageous effects. Besides the clearlyimproved conversion rate and the reduced degradation rate the use ofsaid solvent leads to another important advantage; namely lowerconcentration of bisulfite are used, whereas prior art bisulfitetreatments are performed in the presence of high concentrations ofbisulfite (Fraga and Esteller recommend a final concentration of 5mol/l; supra, p. 642, left column, second paragraph). Such a highconcentration of salt causes a high degradation and leads to problemswithin the subsequent purification and amplification.

A further advantage of the use of n-alkylenglycol compounds as adenaturing agent according to the present invention compared to thealready known denaturing solvents is their higher water solubility. As aconsequence, the reaction compounds, including the scavengers, areapplicable in a broader concentration range. By combining the newsolvents with optimized reaction conditions and new purification methodsthe efficacy of the conversion are further improved, and a sensitive DNAmethylation analysis of tissue or bodily fluids is disclosed andprovided.

In particular embodiments the present invention provides a method forbisulfite conversion of DNA, comprising reacting genomic DNA with abisulfite reagent, wherein said reaction is carried out in the presenceof a compound selected from the group consisting of dioxane, one of itsderivatives, and a similar aliphatic cyclic ether. Preferably, theconcentration of said compounds amounts to about 10-35%, or about20-30%.

Additional embodiments provide a method for bisulfite conversion of DNA,comprising reacting genomic DNA with a bisulfite reagent, wherein saidreaction is carried out in the presence of a compound with the followingformula:

where

n=1-35000

m=1-3

R₁=H, Me, Et, Pr, Bu

R₂=H, Me, Et, Pr, Bu

Preferably, the compound comprises an n-alkylene glycol, a dialkylether, or diethylene glycol dimethyl ether (DME). Preferably, saidcompound is present in a concentration of about 1-35%, or about 5-25%.

Yet additional embodiments provide a method for bisulfite conversion ofDNA, comprising reacting isolated genomic DNA with a bisulfite reagent,wherein the reaction is conducted at a temperature in the range of 0-80°C., and wherein the reaction temperature is briefly increased to a rangeof about 85-100° C. during the course of the conversion. Preferably, thenumber of temperature increases of brief duration amounts to 2 to 5.Preferably, during the temperature increases of brief duration, thereaction temperature increases to about 85 to 100° C. Preferably, thetemperature increases of brief duration increase the reactiontemperature to about 90 to 98° C. Preferably, the converted DNA ispurified via magnetic particles.

Further embodiments provide a method for the bisulfite conversion ofDNA, comprising reacting DNA with a bisulfite reagent, and purifying theconverted DNA by means of ultrafiltration.

Preferably, in the embodiments of the present invention,bisulfite-converted DNA is analyzed, at least in part, by using at leastone method selected from the group consisting of: MSP, HeavyMethyl,MsSNuPE and MethylLight. Preferably, DNA of tissue samples or bodilyfluids is investigated.

Further embodiments provide a method for at least one of diagnosis andprognosis of an adverse event for patients or individuals, comprisinguse of the above described methods, wherein the adverse event is atleast one event selected from the category group consisting of:undesired drug interactions; cancer diseases; CNS malfunctions, damageor disease; symptoms of aggression or behavioral disturbances; clinical,psychological and social consequences of brain damage; psychoticdisturbances and personality disorders; dementia and/or associatedsyndromes; cardiovascular disease, malfunction and damage; malfunction,damage or disease of the gastrointestinal tract; malfunction, damage ordisease of the respiratory system; lesion, inflammation, infection,immunity and/or convalescence; malfunction, damage or disease of thebody as an abnormality in the development process; malfunction, damageor disease of the skin, of the muscles, of the connective tissue or ofthe bones; endocrine and metabolic malfunction, damage or disease;headaches or sexual malfunction.

Additional embodiments provide a method for distinguishing cell types ortissues, or for investigating cell differentiation, comprising use of atleast one of the above-described methods.

Further embodiments provide a kit, comprising: a reagent containingbisulfite; denaturing reagents or solvents, a radical scavenger, andprimers for the production of amplificates. Preferably, the kit furthercomprises at least one of an ultrafiltration tube, and instructions forconducting an assay according to any one of the preceding claims.

DETAILED DESCRIPTION OF THE INVENTION

One embodiment of the present invention provides a method for abisulfite conversion of DNA, wherein DNA is reacted with a bisulfitereagent, characterized in that said reaction is carried out in thepresence of a compound selected from the group consisting of dioxane,one of its derivatives, and a similar aliphatic cyclic ether.

Another embodiment of the present invention provides a method for abisulfite conversion of DNA, wherein DNA is reacted with a bisulfitereagent, characterized in that said reaction is carried out in thepresence of a compound of the following formula:

where

n=1-35000

m=1-3

R₁=H, Me, Et, Pr, Bu

R₂=H, Me, Et, Pr, Bu

Preferred are thus n-alkylene glycol compounds, particularly theirdialkyl ethers, and especially diethylene glycol dimethyl ether (DME).

For both embodiments, the DNA to be investigated may originate fromdifferent sources depending on the diagnostic or scientific objective.For diagnostic investigations, tissue samples are preferably used as theinitial material, but bodily fluids, particularly serum or plasma, canalso be used. It is also possible to use DNA from sputum, stool, urine,or cerebrospinal fluid. Preferably, 5 the DNA is isolated frombiological specimens. The DNA is extracted according to standardmethods, from blood, e.g., with the use of the Qiagen UltraSens™ DNAextraction kit. Other methods for purifying DNA are known to the personskilled in the art.

Subsequently, the isolated DNA may be fragmented, e.g., by reaction withrestriction enzymes. The reaction conditions and the enzymes employedare known to the person skilled in the art and are taken, e.g., from theprotocols supplied by the manufacturers.

The bisulfite conversion may be produced according to the known,protocols indicated above. The reaction may take place both in solutionas well as also on DNA bound to a solid phase. Preferably sodiumdisulfite (=sodium bisulfite/sodium metabisulfite) is used, since it ismore soluble in water than sodium sulfite. The disulfite saltdisproportionates in aqueous solution to the hydrogen sulfite anionsnecessary for the cytosine conversion. When bisulfite concentration isdiscussed below, this refers to the concentration of hydrogen sulfiteand sulfite anions in the reaction solution. For the method according tothe invention, concentration ranges of 0.1 to 6 mol/l are possible (seeabove). Particularly preferred is a concentration range of 1 to 6 mol/l,and most particularly preferred, 2-4 mol/l. However, when dioxane isused, the maximal concentration of bisulfite that can be used is smaller(see below). In selecting the bisulfite concentration, one must considerthat a high concentration of bisulfite leads to a high conversion, butalso leads to a high decomposition rate due to the lower pH.

Dioxane can be utilized in different concentrations. Preferably, thedioxane concentration amounts to 10 to 35% (vol/vol), particularlypreferred is 20 to 30%, and most particularly preferred is 22 to 28%,especially 25%. A dioxane concentration higher than 35% can beproblematic, because this results in a formation of two phases withinthe reaction solution. In the particularly preferred embodiments with adioxane concentration of 22-28%, the final preferred bisulfiteconcentration amounts to 3.3 to 3.6 mol/l, and in the most particularlypreferred embodiment with a dioxane concentration of 25%, it amounts to3.5 mol/l (see Examples herein).

The n-alkylene glycol compounds according to the invention can beutilized in a different concentration range. DME is preferably used inconcentrations between 1-35% (vol/vol). There is preferably between 5and 25%, and most preferably 10% DME.

The preferred scavengers utilized according to the invention arechromane derivatives, e.g., 6-hydroxy-2,5,7,8,-tetramethylchromane2-carboxylic acid (also known as: Trolox-C™). Further scavengers arelisted in the patent application WO 01/98528 (=DE 100 29 915; =U.S.application Ser. No. 10/311,661; incorporated herein by reference in itsentirety).

The bisulfite conversion can be conducted in a wide temperature rangefrom 0 to 95° C. (see above). However, as at higher temperatures therates of both the conversion and decomposition of the DNA increase, in apreferred embodiment the reaction temperature lies between 30-70° C.Particularly preferred is a range between 45-60° C.; most particularlypreferred between 50-55° C. The optimal reaction time of the bisulfitetreatment depends on the reaction temperature. The reaction timenormally amounts to between 1 and 18 hours (see: Grunau et al. 2001,loc. cit.). The reaction time is ordinarily 4-6 hours for a reactiontemperature of 50° C.

In a particularly preferred embodiment of the method according to theinvention, the bisulfite conversion is conducted at mild reactiontemperatures, wherein the reaction temperature is then clearly increasedfor a short time at least once during the course of the conversion. Inthis way, the effectiveness of the bisulfite conversion can besurprisingly clearly be increased. The temperature increases of shortduration are named “thermospikes” below. The “standard” reactiontemperature outside the thermospikes is denoted as the basic reactiontemperature. The basic reaction temperature amounts to between 0 and 80°C., preferably between 30-70° C., most preferably 45°-55° C., asdescribed above. The reaction temperature during a thermospike isincreased to over 85° C. by at least one thermospike. The optimal numberof thermospikes is a function of the basic reaction temperature. Thehigher the optimal number of thermospikes is, the lower is the basicreaction temperature. At least one thermospike is necessary in eachcase. And, on the other hand, in principle, any number of thermospikesis conceivable, f course, it must be considered that with a large numberof temperature increases, the decomposition rate of the DNA alsoincreases, and an optimal conversion is no longer assured. The preferrednumber of thermospikes is thus between 1 and 10 thermospikes each time,depending on the basic reaction temperature. A number of two to 5thermospikes is thus particularly preferred. The thermospikes increasethe reaction temperature preferably to 85 to 100° C., particularlypreferably to 90-98° C., and most preferably to 94° C.-96° C.

The duration in time of the thermospikes also depends on the volume ofthe reaction batch It must be assured that the temperature is increaseduniformly throughout the total reaction solution. For a 20 ul reactionbatch when using a thermocycler a duration between 15 seconds and 1.5minutes, especially a duration between 20 and 50 seconds is preferred.In a particular preferred embodiment the duration is 30 seconds.Operating on a volume of 100 ul the preferred range lies between 30seconds and 5 minutes, especially between 1 and 3 minutes. Particularypreferred are 1.5 minutes. For a volume of 600 ul, a duration of 1 to 6minutes, is preferred, especially between 2 and 4 minutes. Particularlypreferred is a duration of 3 minutes. A person skilled in the art willeasily be able to determine suitable durations of thermospikes inrelation to a variety of reaction volumes.

The above-described use of thermospikes leads to a significantly betterconversion rates in the bisulfite conversion reaction, even when theabove-described denaturing solvents are not utilized. According to theinvention, a method for bisulfite conversion of DNA is herebycharacterized in that the basic reaction temperature amounts to 0° C. to80° C. and that the reaction temperature is increased for a short timeto over 85° C. at least once in the course of the conversion. Theinitial material can be processed as described above.

The preferred temperature ranges, the number of thermospikes, and theirduration correspond to the above-listed ranges. Accordingly, the basicreaction temperature amounts to between 0 and 80° C., preferably between30-70° C., most preferably to 45°-55° C. The reaction temperature isincreased to over 85° C. by at least one thermospike. The preferrednumber of thermospikes is between 1 and 10 thermospikes depending on thebasic reaction temperature. Two to five thermospikes are particularlypreferred. During the thermospikes the reaction temperature increasespreferably to 85 to 100° C., particularly preferably to 90-98° C., andmost preferably to 94° C.-96° C.

The duration in time of the temperature increases also depends on thevolume of the reaction batch (see above).

After the bisulfite conversion is completed, the DNA is desulfonated andpurified. Different methods are known for this purpose (e.g., see: DE101 54 317 A1=U.S. Ser. No. 10/416,624; Grunau et al. 2001, loc. cit.).Normally, the reaction solution is first treated with sodium hydroxide.Subsequently, a neutralization and alcohol precipitation of the DNA arecarried out. In a preferred embodiment of the above-describedembodiments according to the invention, the purification is performed bymeans of a gel filtration, e.g., with Sephadex-G25 columns. Thebisulfite salt can be removed very effectively in this way, without theneed for further washing steps. In a second preferred embodiment, thepurification is conducted via DNA-binding surfaces, e.g., via the WizardDNA purification resin of Promega (see: Kawakami et al., loc. cit.). Athird preferred embodiment utilizes magnetic particles for purification,e.g., with the help of the Magna-Pure process. These purificationmethods lead to particularly good results in combination with then-alkylene glycol compounds according to the invention, particularlywith DME. The purification is conducted according to the manufacturer'sinstructions. It is known to the person skilled in the art that an evenfurther increased yield may be attainable by variation of themanufacturer's instructions by using standard experiments.Correspondingly, optimized protocols are also part of this invention.Further technical instructions for purifying nucleic acids via gelfiltration, DNA-binding surfaces and magnetic particles are known to theperson skilled in the art and are provided, e.g., from themanufacturer's instructions. In a most particularly preferredembodiment, purification is conducted by means of an ultrafiltration.Such a procedure has several technical advantages and results in asurprisingly successful purification of the converted DNA. The recoveryrate of the converted DNA is very high (>85%, see Example 6). This istrue for both, high-molecular DNA as well as also for fragmented DNA,such as found, e.g., in body fluids. The conventional methods forisolating bisulfite-treated DNA, in contrast, only lead to a recoveryrate of approximately 25%. Ultrafiltration also has other advantages.For instance, purification is very flexible with respect to the volumeof the samples to be used. In addition, the bisulfite salts can beremoved almost completely. Furthermore, a desulfonation can be performedon the filter membrane, which additionally results in a savings in time.Different commercially available ultrafiltration systems are known tothe person 5 skilled in the art, which may be used for the methodaccording to the invention. In a preferred embodiment, Microcon™ columnsof Millipore are used. The purification can thus be carried outaccording to a modified manufacturer's protocol. For this purpose, thebisulfite reaction solution is mixed with water and loaded onto theultrafiltration membrane. Subsequently the reaction solution iscentrifuged for about 15 minutes and then washed with 1×TE buffer. TheDNA remains on the membrane in this treatment. Next, desulfonation isperformed. For this purpose, 0.2 mol/l NaOH is added and the DNAincubated for 10 min. Another centrifugation (10 min) is then conducted,followed by a washing step with 1×TE buffer. The DNA is subsequentlyeluted. For this purpose, the membrane is mixed with 50 ul of warm 1×TEbuffer (50° C.) for 10 minutes. The membrane is turned over according tothe manufacturer's instructions and a repeated centrifugation isconducted, by means of which the DNA is removed from the membrane. Nowthe eluate can be used directly for the intended detection reactions. Itis known to the person skilled in the art that other procedures may beindicated with other ultrafiltration systems, and that a good yield canalso be obtained by varying the above-indicated conditions. Thecorresponding embodiments are also part of this invention.

The above-described use of ultrafiltration also facilitates a clearlyimproved purification of bisulfite-converted DNA, when theabove-described denaturing solvents are not utilized or when conversionis conducted without thermospikes. Therefore, according to theinvention, a method for the bisulfite conversion of DNA is herebycharacterized in that the purification of the converted DNA takes placeby means of ultrafiltration. The initial material can thus be processedup as described above. Thermospikes may also be utilized. The preferredtemperature ranges, the number of thermospikes, and their durationcorrespond to the above-listed ranges (see above). Also, ultrafiltrationis preferably conducted as described above. Accordingly, differentultrafiltration systems may be utilized. In a preferred embodiment, theMicrocon™ columns of Millipore are used. The purification is preferablyconducted as described above according to a modified manufacturer'sprotocol. It is known to the person skilled in the art that otherprocedures can be indicated with other ultrafiltration systems, and thatan even further improved yield can also be obtained by varying theabove-indicated conditions, he corresponding embodiments are also partof this invention.

The DNA which has been converted and purified via the above-describeddifferent embodiments may now be analyzed in different ways. It isparticularly preferred to amplify the DNA first by means of a polymerasechain reaction. Thus, a selective amplification of the originallymethylated or unmethylated DNA can be assured via different methods,e.g., via the so-called “Heavy-Methyl” method for review see. Cottrellet al.; A real-time PCR assay for DNA-methylation usingmethylation-specific blockers. Nucleic Acids Res., 32:e10, 2004. WO02/072880.) or the so-called “methylation-sensitive PCR” (“MSP”; see:Herman et al.: Methylation-specific PCR: a novel PCR assay formethylation status of CpG islands. Proc Natl Acad Sci USA. 93:9821-6,1996). The amplificates obtained may be detected via conventionalmethods, e.g., via primer extension reactions (“MsSNuPE”; see, e.g., DE100 10 280=U.S. Ser. No. 10/220,090) or via hybridization to oligomerarrays (see e.g.: Adorjan et al., Tumour class prediction and discoveryby microarray-based DNA methylation analysis. Nucleic Acids Res.,30:e21, 2002). In another particularly preferred embodiment, theamplificates are analyzed with the use of PCR Real Time variants (see:U.S. Pat. No. 6,331,393 “Methyl Light”). Preferred variants aretherefore the “Taqman” and the “Lightcycler” methods.

The methods disclosed here are preferably used for the diagnosis and/orprognosis of adverse events for patients or individuals, whereby theseadverse events belong to at least one of the following categories:undesired drug interactions; cancer diseases; CNS malfunctions, damageor disease; symptoms of aggression or behavioral disturbances; clinical,psychological and social consequences of brain damage; psychoticdisturbances and personality disorders; dementia and/or associatedsyndromes; cardiovascular disease, malfunction and damage; malfunction,damage or disease of the gastrointestinal tract; malfunction, damage ordisease of the respiratory system; lesion, inflammation, infection,immunity and/or convalescence; malfunction, damage or disease of thebody as an abnormality in the development process; malfunction, damageor disease of the skin, of the muscles, of the connective tissue or ofthe bones; endocrine and metabolic malfunction, damage or disease;headaches or sexual malfunction.

The new method also serves in a particularly preferred manner fordistinguishing cell types, tissues or for investigating celldifferentiation.

The new method also serves in a particularly preferred manner foranalyzing the response of a patient to a drug treatment.

The subject of the present invention is also a kit, which contains areagent containing bisulfite, denaturing reagents or solvents, as wellas scavengers, primers for the production of the amplificates as wellas, optionally, an ultrafiltration tube or instructions for conductingan assay.

EXAMPLES

The following non-limiting examples facilitate disclosure of aspects ofthe broader invention.

Example 1 Automation of the Bisulfite Reaction

The application of the method for detecting the methylation state ofcytosines in the factor VIII gene of a genomic DNA sample, which istreated with a restriction endonuclease according to the instructions ofthe manufacturer, is described in the present example. The method isbased on the use of an automatic pipetting system (MWG RoboSeq 4204)with four separate vertically movable adapters for exchangeablepipetting tips, so as to exclude cross contaminations. The pipettingsystem makes possible the pipetting of 100 ul [aliquots] with an errorof less than +2 ul. The operating plate of the automatic pipettingsystem is equipped with six racks for pipetting tips and eight 2pipetting positions, two of which can be cooled, a reagent rack that canbe cooled, a stacking system for 10 microtiter plates, a pipette tipwashing station and a device for separating the pipette tips from theadapter.

The automatic pipetting system is connected to a computer by means of aserial interface and is controlled by means of a software program, whichpermits the free programming of all pipetting steps necessary for theapplication of the method.

In the first step of the method, an aliquot of the DNA sample ispipetted by hand into one of the 96 freely selectable positions of amicrotiter plate. The microtiter plate is then subsequently heated to96° C. with the use of an Eppendorf MasterCycler for denaturing thepretreated DNA sample. The microtiter plate is then transferred to theautomatic pipetting system. Aliquots of a denaturing agent (dioxane), a3.3 M sodium hydrogen sulfite solution, and a solution of a scavenger inthe denaturing agent used are pipetted one after the other in aprogram-controlled manner from the reagent rack into all positions thatcontain DNA. Then the microtiter plate is incubated in the EppendorfMastercycler, so that all unmethylated cytosine residues in the DNAsample are converted into a bisulfite adduct with the action of thesodium hydrogen sulfite.

After the bisulfite treatment, the microtiter plate is transferred fromthe thermocycler to the automatic pipetting system. A second microtiterplate of the same type is then positioned. First, a basic Tris-HClbuffer (pH 9.5) and then an aliquot of the bisulfite-treated DNA aretransferred into the corresponding positions of the second microtiterplate in all chambers whose equivalent positions on the first microtiterplate contain a bisulfite-treated DNA sample. The bisulfite adducts ofthe unmethylated cytosine residues are converted to uracil residues inthe basic solution.

The targeted amplification of one strand (the sense strand in thepresent example) of the bisulfite-treated DNA is conducted by apolymerase chain reaction (PCR). A pair of primers of type I (AGG GAGTTT TTT TTA GGG AAT AGA GGG A (SEQ. ID: 1) and TAA TCC CAA AAC CTC TCCACT ACA ACA A (SEQ ID: 2) are used, which permit the specificamplification of a successfully bisulfite-treated DNA strand, but not aDNA strand, whose unmethylated cytosine residues were not converted touracil residues or were incompletely converted. A third microtiter plateof the same type is positioned in the automatic pipetting system for thePCR reaction. In all chambers, whose equivalent positions on the firstmicrotiter plate contain a bisulfite-treated DNA sample, an aliquot of astock solution, which contains a PCR buffer, a DNA polymerase and aprimer of type 1 is first automatically pipetted. Then, an aliquot ofthe diluted bisulfite-treated DNA is transferred automatically from eachposition of the second microtiter plate to the corresponding position ofthe third microtiter plate, before the latter is transferred to thecycler for conducting the PCR reaction. The PCR product is identified byagarose gel electrophoresis and subsequent staining with ethidiumbromide (FIG. 1). FIG. 1 shows the gel image of a PCR-amplifiedbisulfite-treated DNA strand (left: molecular weight marker, right: PCRproduct).

Example 2 Optimized Bisulfite Conversion by Addition of Dioxane for theDetection of DNA in Plasma Samples

The inventive optimized bisulfite method makes possible a sensitivemethylation analysis of DNA obtained from body fluids. For example, 1 mlof human plasma was mixed with a specific quantity of human DNA. The DNAwas isolated from the plasma samples via the Magna Pure method (Roche)according to the manufacturer's instructions. The 100 ul of eluateresulting from the purification were utilized completely in thefollowing bisulfite reaction. The conversion according to a standardmethod (Frommer et al., loc. cit.) was conducted as a control. Theprocedure for the method according to the invention was as follows: Theeluate was mixed with 354 ul of bisulfite solution (5.89 mol/l) and 146ul of dioxane containing a radical scavenger(6-hydroxy-2,5,7,8-tetramethylchromane 2-carboxylic acid, 98.6 mg in 2.5ml of dioxane). The reaction mixture was denatured for 3 min at 99° C.and subsequently incubated with the following temperature program for atotal of 5 h: 30 min 50° C.; one thermospike (99.9° C.) for 3 min; 1.5 h50° C.; one thermospike (99.9° C.) for 3 min; 3 h 50° C. The reactionmixtures of both the control as well as also of the method according tothe invention were subsequently purified by ultrafiltration by means ofa Millipore Microcon™ column. The purification was conducted essentiallyaccording to the manufacturer's instructions. For this purpose, thereaction mixture was mixed with 300 ul of water, loaded onto theultrafiltration membrane, centrifuged for 15 min and subsequently washedwith 1×TE buffer. The DNA remains on the membrane in this treatment.Then desulfonation is performed. For this purpose, 0.2 mol/l NaOH wasadded and incubated for 10 min. A centrifugation (10 min) was thenconducted, followed by a washing step with 1×TE buffer. After this, theDNA was eluted. For this purpose, the membrane was mixed for 10 minuteswith 50 ul of warm 1×TE buffer (50° C.). The membrane was turned overaccording to the manufacturer's instructions. Subsequently a repeatedcentrifugation was conducted, with which the DNA was removed from themembrane. 10 ul of the eluate were utilized for the followingLightcycler™ Real Time PCR. A region of the human beta-actin gene wasanalysed (see Miyamoto: Nucleotide sequence of the human beta-actinpromoter 5′ flanking region; Nucleic Acids Res. 15:9095, 1987)). Thefollowing primer and probes were used: Forward primer: TGG TGA TGG AGGAGG TTT AGT AAG T (SEQ ID 3); reverse primer: AAC CAA TAA AAC CTA CTCCTC CCT TAA (SEQ ID 4); donor probe: TTG TGA ATT TGT GTT TGT TAT TGT GTGTTG-flou (SEQ ID 5); acceptor probe: LC Red640-TGG TGG TTA TTT TTT TTATTA GGT TGT GGT-Phos (SEQ ID 6). The amplification was conducted bymeans of a bisulfite-specific assay. The fluorescent signals weredetected and calculated with the Lightcycle software. The amount ofconverted and isolated DNA could be quantified by a comparison withcalibration curves. The optimized method produced a DNA concentration of133.21 ng/100 ul, while the conventional method led to a concentrationof 41.03 ng/100 ul. The method according to the invention thus madepossible a yield that was three times higher than the conventionalmethod.

Example 3 Optimized Bisulfite Conversion by Addition of DME for theDetection of DNA in Plasma Samples

It will be shown that the optimized bisulfite method makes possible asensitive methylation analysis of DNA obtained from body fluids. Forthis purpose, 1 ml of human plasma was mixed with a specific quantity ofhuman DNA. The DNA was isolated from the plasma samples via the MagnaPure method (Roche) according to the manufacturer's instructions. The100 ul of eluate resulting from the purification were utilizedcompletely in the following bisulfite reaction. Conversion according toa standard method (Frommer et al., loc. cit.) was conducted as acontrol. The procedure for the method according to the invention was asfollows: The eluate was mixed with 354 ul of bisulfite solution (5.89mol/l) and 46 ul of DME containing a radical scavenger(6-hydroxy-2,5,7,8-tetramethylchromane 2-carboxylic acid, 98.6 mg in 787ul of DME). The reaction mixture was denatured for 3 min at 99° C. andsubsequently incubated with the following temperature program for atotal of 5 h: 30 min 50° C.; one thermospike (99.9° C.) for 3 min; 1.5 h50° C.; one thermos-pike (99.9° C.) for 3 min; 3 h 50° C. The reactionmixtures of both the control as well as also of the method according tothe invention were subsequently purified by ultrafiltration by means ofa Millipore Microcon™ column. The purification was conducted essentiallyaccording to the manufacturer's instructions. For this purpose, thereaction mixture was mixed with 300 ul of water, loaded onto theultrafiltration membrane, centrifuged for 15 min and subsequently washedwith 1×TE buffer. The DNA remains on the membrane in this treatment.Then desulfonation is performed. For this purpose, 0.2 mol/l NaOH wasadded and incubated for 10 min. A centrifugation (10 min) was thenconducted, followed by a washing step with 1×TE buffer. After this, theDNA was eluted. For this purpose, the membrane was mixed for 10 minuteswith 50 ul of warm 1×TE buffer (50° C.). The membrane was turned overaccording to the manufacturer's instructions. Subsequently a repeatedcentrifugation was conducted, by means of which the DNA was removed fromthe membrane. 10 ul of the eluate were utilized for the followingLightcycler Real Time PCR. A region of the human beta-actin gene wasanalyzed by using the primers and probes described in example 2. Theamplification was conducted by means of a bisulfite-specific assay. Theresults calculated by the Lightcycler software are shown in FIG. 2. Thecurves on the left correspond to the optimized method, while the curveson the right correspond to the conventional method. It is shown that theoptimized method produces a significant fluorescent signal, even with asmall number of cycles. The DNA yield is thus higher than with theconventional method. The amount of converted DNA can be quantified by acomparison with calibration curves. The optimized method produced a DNAconcentration of 133.27 ng/100 ul, while the conventional method led toa concentration of 41.03 ng/100 ul. The method according to theinvention thus made possible a yield that was three times higher thanthe conventional method.

Example 4 Bisulfite Conversion with Help of Thermospikes

2 ul of ddH₂0 were added to 1 ul of highly pure human DNA digested withMssl (Promega; 160 ng). The samples were denatured for 10 minutes at 96°C. Then roughly 10 ul of bisulfite solution (5.85 mol/l) and 7 ul of aradical scavenger/dioxane mixture (5 ul of dioxane plus 2 ul ofscavenger) were added. After this, the first sample (0 h value) wasremoved and placed on ice. The reaction mixture was incubated for 30seconds at 96° C. and subsequently for 59.5 minutes at 50° C. The secondsample (1 h value) was removed and placed on ice. The third sample (2 hvalue) was incubated once more for 30 seconds at 96° C. and for 59.5minutes at 50° C. Subsequently this sample was also placed on ice. Thefourth sample (3 h value) was incubated once more for 30 seconds at 96°C. and for 59.5 minutes at 50° C., and subsequently also cooled. 30 ulof ddH₂0 were added to the samples. The reaction mixture was purifiedvia G25 Sephadex columns. The eluate was mixed with 50 ul of 100 mmol/lTris-HCl (pH 9.5) and desulfonated at 96° C. for 20 minutes. 2 ul ofthis solution were used for each PCR reaction. Two bisulfite-specificfragments, two nonspecific fragments and a genomic fragment wereamplified each time in the PCR. The bisulfite-specific fragments aremore intensely amplified, the further the bisulfite conversion hasprogressed. The nonspecific fragments are amplified independently of thebisulfite conversion and provide an indication of the degradation of theDNA. The genomic fragment is amplified only insofar as genomic DNA whichis not converted with bisulfite is still present. The amplification ofthe genomic DNA is thus a measurement for an incomplete bisulfiteconversion. The amplificates separated in agarose gels can be seen inFIG. 3. It is shown that in the method according to the invention, alarge part of the DNA has been converted even after one hour. GenomicDNA can no longer be detected after three hours at the latest, i.e., thebisulfite conversion is complete. In conventional bisulfite treatment,corresponding values are produced at the earliest after 5 h (see below).

Example 5 Comparison of Bisulfite Treatment with Thermospikes toBisulfite Treatment without Thermospikes

Samples which were treated as in Example 4 were incubated with areaction time of 3 or 5 h with two thermospikes. The controls werereacted without thermospikes. Purification and PCR were conducted asdescribed above. Two bisulfite-specific fragments were amplified. One ofthe fragments was cytosine-rich. A relatively long reaction time wasthus necessary in order to attain a complete conversion. The otherfragment, in contrast, was cytosine-poor and was thus completelyconverted after a relatively short time. The results of theamplifications are shown in FIG. 4. The conventional bisulfiteconversion can be seen in the figures on the left, while the optimizedconversion with thermospikes can be seen in the figures on the right.The cytosine-rich fragment is plotted on the left each time, while thecytosine-poor fragment is plotted on the right. It is shown that themethod according to the invention makes possible a clearly moresensitive detection. Therefore, the cytosine-rich fragment can beclearly detected even after 3 hours with a thermospike treatment, whileit cannot be detected with the conventional method even after 5 hours ofreaction time.

Example 6 DNA Recovery Rate in the Method According to the Invention

It will be shown that the method according to the invention makespossible a very effective bisulfite conversion and purification. Forthis purpose, different amounts of M13-DNA and human DNA were dissolvedin 100 ul of water. The DNA solutions were mixed with 354 ul ofbisulfite solution (5.89 mol/l) and 146 ul of dioxane containing aradical scavenger (6-hydroxy-2,5,7,8-tetramethylchromane 2-carboxylicacid, 98.6 mg in 2.5 ml of dioxane). The reaction mixture was denaturedfor 3 min at 99° C. and subsequently incubated with the followingtemperature program for a total of 5 h: 30 min 50° C.; one thermospike(99.9° C.) for 3 min; 1.5 h 50° C.; one thermospike (99.9° C.) for 3min; 3 h 50° C. The reaction mixtures were subsequently purified byultrafiltration by means of a Millipore Microcon™ column. Thepurification was conducted essentially according to the manufacturer'sinstructions. For this purpose, the reaction mixture was mixed with 300μl of water, loaded onto the ultrafiltration membrane, centrifuged for15 min and subsequently washed with 1×TE buffer. The DNA remains on themembrane in this treatment. Then desulfonation is performed. For thispurpose, 0.2 mol/l NaOH was added and incubated for 10 min. Acentrifugation (10 min) was then conducted, followed by a washing stepwith 1×TE buffer. After this, the DNA was eluted. For this purpose, themembrane was mixed for 10 minutes with 50 ul of warm 1×TE buffer (50°C.). The membrane was turned over according to the manufacturer'sinstructions. Subsequently a repeated centrifugation was conducted, bymeans of which the DNA was removed from the membrane. The DNAconcentrations were then determined fluorometrically (olive green). Thedata are shown in Table 1. The recovery rate of the DNA amounts to atleast 75%. In the known methods for bisulfite conversion andpurification of DNA, the recovery rates, in contrast, lie below 25%.

TABLE 1 Utilized amount M 13 DNA Human single-stranded DNA of DNA in ngafter ultrafiltration after ultrafiltration 6000 not determined 108.5%4000 not determined 87.1% 2000 86.91% 84.8% 1000 90.54% 81.51% 20092.69% 91.59% 100 96.77% 74.23%

1. A method for bisulfite conversion of DNA, comprising reacting genomicDNA with a bisulfite reagent, wherein the reaction is carried out in thepresence of a compound having the following formula:


2. The method of claim 1, wherein the compound comprises an n-alkyleneglycol.
 3. The method of claim 2, wherein the compound comprises adialkyl ether.
 4. The method of claim 3, wherein the compound comprisesdiethylene glycol dimethyl ether (DME).
 5. The method of claim 1,wherein the compound is present in a concentration of about 1-35%. 6.The method of claim 1, wherein the compound is present in aconcentration of about 5-25%.
 7. The method of claim 1, wherein theconverted DNA is analyzed using a method selected from the groupconsisting of: MSP, HeavyMethyl, MsSNuPE and MethylLight.
 8. The methodof claim 1, wherein DNA of tissue samples or bodily fluids isinvestigated.
 9. A method for at least one of diagnosis and prognosis ofan adverse event for patients or individuals, comprising use of themethod of claim 1, wherein the adverse event is at least one eventselected from the category group consisting of: undesired druginteractions; cancer diseases; CNS malfunctions, damage or disease;symptoms of aggression or behavioral disturbances; clinical,psychological and social consequences of brain damage; psychoticdisturbances and personality disorders; dementia and/or associatedsyndromes; cardiovascular disease, malfunction and damage; malfunction,damage or disease of the gastrointestinal tract; malfunction, damage ordisease of the respiratory system; lesion, inflammation, infection,immunity and/or convalescence; malfunction, damage or disease of thebody as an abnormality in the development process; malfunction, damageor disease of the skin, of the muscles, of the connective tissue or ofthe bones; endocrine and metabolic malfunction, damage or disease;headaches or sexual malfunction.
 10. A method for distinguishing celltypes or tissues, or for investigating cell differentiation, comprisinguse of the method of claim 1.